pT7-7 cloning

Your "pT7-7 problem" is most likely related to the design of your PCR
primers. NdeI digestion requires quite a bit of an overhang on either side
of CATATG-palindrome. Recognition site provides for recognition but not
necessarily is sufficient for the enzyme attachement. NEB catalog (1995)
has a good table (p208) of a chain length required for the digestion with
different enzymes. It will provide you with a reference point.
When I design my primers for the NdeI-add-on-PCR I provide for at least 12
nucleotides of some nonsense sticking out in front of the site.
Good luck
PS: If you happened to have the sequence of pT7-7 I would appreciate your
sharing it with me (by e-mail). We have the vector itself but do not have
the sequence outside the cloning cassette.
Thank you
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Michael J. Tuvim
Research Assistant Professor, Dept.of Medicine, Baylor College of Medicine.
VAMC, bldg 109, rm 106, 2002 Holcombe blvd, Houston, TX, 77030
Tel: 713/794-7794(5), FAX: 713/794-7853, e-mail: mtuvin at bcm.tmc.edu
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